Quadratic recycling amplification for label-free and sensitive visual detection of HIV DNA

• Sensitive and visual detection of HIV DNA down to the low picomolar level is realized by a new quadratic signal amplification strategy.
• Quadratic signal amplification is achieved by two exonuclease III-assisted DNA recycling cycles.
• The employment of the un-modified, G-quadruplex sequence-integrated hairpin DNA probes leads to label-free detection of HIV DNA.
• The proposed method can be applied for visual detection of HIV DNA in diluted serum samples.

Visual detections have attracted great research attentions recently due to their convenient monitoring of the target analytes without using any advanced instruments. However, achieving visual detection of trace amounts of biomolecules with PCR-like sensitivity remains a major challenge. In current work, we describe a new quadratic signal amplification strategy for sensitive visual detection of HIV DNA biomarkers based on exonuclease III (Exo III)-assisted DNA recycling amplification and DNAzymes. The presence of the target HIV DNA leads to two independent and simultaneous DNA recycling processes to achieve quadratic signal amplification with the assistance of Exo III. This quadratic signal amplification results in catalytic cleavage of the G-quadruplex sequence-locked hairpin probes to release numerous active G-quadruplex sequences, which further associate with hemin to form DNAzymes and cause significantly intensified color change for sensitive and visual detection of HIV DNA down to 2.5 pM. The proposed visual detection method employs un-modified hairpin DNA as probes, avoids using any complex and expensive instruments for signal transduction and is essentially simple. This method also shows single-base mismatch discrimination capability as well. All these features make our developed DNA detection method holds great potential for visual monitoring of various DNA biomarkers at ultralow levels with careful and proper probe designs.